Terminal CCA Research Articles

Mutations in the peptidyl transferase region of E. coli 23S rRNA affecting translational accuracy.

We have produced mutations in a cloned Escherichia coli 23S rRNA gene at positions G2252 and G2253. These sites are protected in chemical footprinting studies by the 3' terminal CCA of P site-bound tRNA. Three possible base changes were introduced at each position and the mutations produced a range of effects on growth rate and translational accuracy. Growth of cells bearing mutations at 2252 was severely compromised while the only mutation at 2253 causing a marked reduction in growth rate was a G to C transversion. Most of the mutations affected translational accuracy, causing increased readthrough of UGA, UAG and UAA nonsense mutations as well as +1 and -1 frameshifting in a lacZ reporter gene in vivo. C2253 was shown to act as a suppressor of a UGA nonsense mutation at codon 243 of the trpA gene. The C2253 mutation was also found not to interact with alleles of rpsL coding for restrictive forms of ribosomal protein S12. These results provide further evidence that nucleotides localized to the P site in the 50S ribosomal subunit influence the accuracy of decoding in the ribosomal A site.

Role of the D arm and the anticodon arm in tRNA recognition by eubacterial and eukaryotic RNase P enzymes.

Truncated precursor tRNAs lacking the D arm or anticodon arm were studied in vitro as substrates for RNase P enzymes from Escherichia coli, Thermus thermophilus (eubacteria), and HeLa. Deletion of the D arm still allowed 5'-processing by E. coli RNase P, but strongly impaired maturation by T. thermophilus and HeLa extracts. In contrast, deletion of the anticodon arm had no influence on processing by RNase P activities from all three organisms. Inhibition kinetics and gel retardation studies showed that deletion of the D arm leads to low-affinity binding to E. coli RNase P RNA (M1 RNA). However, the E. coli enzyme appears to form sufficiently strong contacts in the region of the T arm, acceptor stem, and CCA terminus to still allow productive enzyme-substrate interaction even in the absence of the structural contribution provided by the D arm. Pb(2+)-induced hydrolysis of a tRNAGly from T. thermophilus gave identical cleavage patterns in the D arm and anticodon loop in the absence and presence of E. coli M1 RNA, whereas lead hydrolysis was strongly reduced at the CUCCAA 3'-terminus due to the presence of the enzyme.

Substitution of the 3' terminal adenosine residue of transfer RNA in vivo.

We have altered by site-directed mutagenesis the 3' terminal adenosine residue of a tRNA(Tyrsu3+) gene encoded on a single-copy plasmid and examined the consequences of these substitutions on suppressor activity in vivo. Our data show that mutant su3 genes containing 3'-CCC, -CCG, or -CCU termini instead of -CCA can be efficiently transcribed and processed in Escherichia coli to generate functional suppressor tRNAs. However, in contrast to normal tRNA genes, both tRNA nucleotidyltransferase and exoribonuclease activities are required to obtain suppression by the mutant tRNAs, indicating that removal of the incorrect 3' terminal residue and resynthesis of the normal -CCA terminus are occurring in this situation. In addition, a low level of suppressor activity and tRNA repair was found in cells devoid of tRNA nucleotidyltransferase, suggesting that an additional activity able to partially repair the 3' end of tRNA is present in E. coli. The use of mutant strains lacking one or several exoribonucleases revealed that the various RNAses have very different specificities for removal of incorrect 3' residues and that these differ greatly from their action on CCA-ending tRNA. These data show that the 3' terminal adenosine residue is necessary for tRNA function in vivo and that cells can compensate for its alteration by changes in the normal pathway of tRNA metabolism.

Two Streptomyces lividans 66 transfer RNA genes with anticodons corresponding to serine (AGC) and arginine (CGU) codons.

From the nucleotide sequence of a 2110-bp Streptomyces lividans 66 DNA fragment two transfer RNA genes were identified: tRNA(Ser) (AGC) and tRNA(Arg) (CGU). These tRNA genes are transcribed from the same DNA strand and both are preceded by a putative promoter structure. They are separated by an intergenic region of 191 bp. Like most Streptomyces tRNA genes described so far, they do not encode the 3' terminal CCA of mature tRNAs. Both genes are followed by extensive inverted repeats, which could serve as transcriptional terminator signals. Remarkably, these hairpin structures share an identical 9 base pair stretch (5'-GAAGCCCCG-3'). Furthermore, the tRNA(Arg) region is followed by two potential open reading frames, which are encoded on complementary strands and have 3' overlapping ends. The gene for tRNA(Arg) (CGU) is the first Streptomyces tRNA gene described so far which encodes the translation of a codon with uridine at the third (wobble) position.

Two Streptomyces lividans 66 transfer RNA genes with anticodons corresponding to serine (AGC) and arginine (CGU) codons

From the nucleotide sequence of a 2110-bp Streptomyces lividans 66 DNA fragment two transfer RNA genes were identified: tRNA(Ser) (AGC) and tRNA(Arg) (CGU). These tRNA genes are transcribed from the same DNA strand and both are preceded by a putative promoter structure. They are separated by an intergenic region of 191 bp. Like most Streptomyces tRNA genes described so far, they do not encode the 3' terminal CCA of mature tRNAs. Both genes are followed by extensive inverted repeats, which could serve as transcriptional terminator signals. Remarkably, these hairpin structures share an identical 9 base pair stretch (5'-GAAGCCCCG-3'). Furthermore, the tRNA(Arg) region is followed by two potential open reading frames, which are encoded on complementary strands and have 3' overlapping ends. The gene for tRNA(Arg) (CGU) is the first Streptomyces tRNA gene described so far which encodes the translation of a codon with uridine at the third (wobble) position.

A mutation in the tRNA nucleotidyltransferase gene promotes stabilization of mRNAs in Saccharomyces cerevisiae.

To identify trans-acting factors involved in mRNA decay in the yeast Saccharomyces cerevisiae, we have begun to characterize conditional lethal mutants that affect mRNA steady-state levels. A screen of a collection of temperature-sensitive mutants identified ts352, a mutant that accumulated moderately stable and unstable mRNAs after a shift from 23 to 37 degrees C (M. Aebi, G. Kirchner, J.-Y. Chen, U. Vijayraghavan, A. Jacobson, N.C. Martin, and J. Abelson, J. Biol. Chem. 265:16216-16220, 1990). ts352 has a defect in the CCA1 gene, which codes for tRNA nucleotidyltransferase, the enzyme that adds 3' CCA termini to tRNAs (Aebi et al., J. Biol. Chem., 1990). In a shift to the nonpermissive temperature, ts352 (cca1-1) cells rapidly cease protein synthesis, reduce the rates of degradation of the CDC4, TCM1, and PAB1 mRNAs three- to fivefold, and increase the relative number of ribosomes associated with mRNAs and the overall size of polysomes. These results were analogous to those observed for cycloheximide-treated cells and are generally consistent with models that invoke a role for translational elongation in the process of mRNA turnover.

A mutation in the tRNA nucleotidyltransferase gene promotes stabilization of mRNAs in Saccharomyces cerevisiae.

To identify trans-acting factors involved in mRNA decay in the yeast Saccharomyces cerevisiae, we have begun to characterize conditional lethal mutants that affect mRNA steady-state levels. A screen of a collection of temperature-sensitive mutants identified ts352, a mutant that accumulated moderately stable and unstable mRNAs after a shift from 23 to 37 degrees C (M. Aebi, G. Kirchner, J.-Y. Chen, U. Vijayraghavan, A. Jacobson, N.C. Martin, and J. Abelson, J. Biol. Chem. 265:16216-16220, 1990). ts352 has a defect in the CCA1 gene, which codes for tRNA nucleotidyltransferase, the enzyme that adds 3' CCA termini to tRNAs (Aebi et al., J. Biol. Chem., 1990). In a shift to the nonpermissive temperature, ts352 (cca1-1) cells rapidly cease protein synthesis, reduce the rates of degradation of the CDC4, TCM1, and PAB1 mRNAs three- to fivefold, and increase the relative number of ribosomes associated with mRNAs and the overall size of polysomes. These results were analogous to those observed for cycloheximide-treated cells and are generally consistent with models that invoke a role for translational elongation in the process of mRNA turnover.

Transfer RNA genes from the hyperthermophilic archaeon, Methanopyrus kandleri

Genes encoding the Leu (GAG), Ser (UGA), Gln (UUG) and Lys (UUU) tRNAs have been cloned and sequenced from the deep sea hyperthermophilic Archaeon, Methanopyrus kandleri. Sequences conforming to the TATA box element established for methanogen promoters are located upstream of the tRNA Gln and tRNA Lys genes. All four of the tRNA genes appear to encode the 3′ terminal CCA residues of the mature tRNA. These methanogen tRNAs are predicted to contain most, but not all, invariant residues and are characterized by a high level of G + C base pairing, consistent with the 98°C optimum growth temperature of M. kandleri.

Specificity of human immunodeficiency virus-1 reverse transcriptase-associated ribonuclease H in removal of the minus-strand primer, tRNA(Lys3).

We have examined the specificity of human immunodeficiency virus-1 (HIV-1) reverse transcriptase-associated RNase H in removing the tRNA(Lys3) (-)-strand primer in vitro using a model substrate. This substrate represents an intermediate in the reverse transcription process where the tRNA(Lys3) primer has not yet been removed after (+)-strand strong stop DNA synthesis. The substrate consists of an RNA oligonucleotide corresponding to the 3'-terminal 17 nucleotides of the tRNA(Lys3) linked to U5 DNA and annealed to single-stranded DNA containing the U5 and the primer-binding site. Upon incubation with HIV-1 reverse transcriptase p66/p51 heterodimer, the minus-strand DNA product resulting from RNase H cleavage retained the 3'-rA from the model tRNA primer. Changing the 3'-terminal AMP of the model tRNA primer from rA to dA did not alter the RNase H cleavage site. Further, the retention of AMP was not dependent on recognition of adjacent U5 sequences or the CCA terminus of the model tRNA(Lys3). The synthetic RNA primer was released as an intact species by a single endonucleolytic cleavage 5' of the rA. The cleavage patterns of Moloney murine leukemia virus and avian myoblastosis virus RNase H activities on the HIV-1 model substrate were more heterogeneous compared to HIV-1 RNase H. This specificity of HIV-1 RNase H would result in linear DNA molecules with a single rA at the U5 terminus and would provide two bases adjacent to the conserved CA dinucleotide to be cleaved away during the integration process.

Important 2′-hydroxyl groups in model substrates for M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli

The role of 2′-hydroxyl groups in a model substrate for RNase P from Escherichia coli was studied using mixed DNA/RNA derivatives of such a substrate. The presence of the 2′-hydroxyl groups of nucleotides at positions −1 and −2 in the leader sequence and at position 1, as well as at the first C in the 3′-terminal CCA sequence, are important but not absolutely essential for efficient cleavage of the substrate by RNase P or its catalytic RNA subunit, M1 RNA. The 2′-hydroxyl groups in the substrate that are important for efficient cleavage also participate in the binding of Mg 2+. An all-DNA external guide sequence (EGS) can efficiently render a potential substrate, derived from the model substrate, susceptible to cleavage by the enzyme or its catalytic RNA subunit. Furthermore, both DNA and RNA EGSs turn over during the reaction with RNase P in vitro. The identity of the nucleotide at position 1 in the substrate, the adjacent Mg 2+-binding site in the leader sequence, and the junction of the single and double-stranded regions are the important elements in the recognition of model substrates, as well as in the identification of the sites of cleavage in those model substrates.

Cleavage Specificity of Chloroplast and Nuclear tRNA 3′-Processing Nucleases

tRNAs in eukaryotic nuclei and organelles are synthesized as precursors lacking the 3'-terminal CCA sequence and possessing 5' (leader) and 3' (trailer) extensions. Nucleolytic cleavage of the 3' trailer and addition of CCA are therefore required for formation of functional tRNA 3' termini. Many chloroplast tRNA genes encode a C at position 74 which is not removed during processing but which can be incorporated as the first base of the CCAOH terminus. Sequences downstream of nucleotide 74, however, are always removed. Synthetic yeast pre-tRNA(Phe) substrates containing the complete CCA74-76 sequence were processed with crude or partially purified chloroplast enzyme fractions. The 3'-extended substrates (tRNA-CCA-trailer) were cleaved exclusively between nucleotides 74 and 75 to give tRNA-COH, whereas a 3'-mature transcript (tRNA-CCAOH) was not cleaved at all. A 5'-, 3'-extended chloroplast tRNA-CAG-trailer was also processed entirely to tRNA-COH. Furthermore, a 5'-mature, 3'-extended yeast pre-tRNA(Phe) derivative, tRNA-ACA-trailer, in which C74 was replaced by A, was cleaved precisely after A74. In contrast, we found that a partially purified enzyme fraction (a nuclear/cytoplasmic activity) from wheat embryo cleaved the 3'-extended yeast tRNA(Phe) precursors between nucleotides 73 and 74 to give tRNA(OH). This specificity is consistent with that of all previously characterized nuclear enzyme preparations. We conclude that (i) chloroplast tRNA 3'-processing endonuclease cleaves after nucleotide 74 regardless of the nature of the surrounding sequences; (ii) this specificity differs from that of the plant nuclear/cytoplasmic processing nuclease, which cleaves after base 73; and (iii) since 3'-mature tRNA is not a substrate for either activity, these 3' nucleases must require substrates possessing a 3'-terminal extension that extends past nucleotide 76. This substrate specificity may prevent mature tRNA from counterproductive cleavage by the 3' processing system.

Cleavage specificity of chloroplast and nuclear tRNA 3'-processing nucleases.

tRNAs in eukaryotic nuclei and organelles are synthesized as precursors lacking the 3'-terminal CCA sequence and possessing 5' (leader) and 3' (trailer) extensions. Nucleolytic cleavage of the 3' trailer and addition of CCA are therefore required for formation of functional tRNA 3' termini. Many chloroplast tRNA genes encode a C at position 74 which is not removed during processing but which can be incorporated as the first base of the CCAOH terminus. Sequences downstream of nucleotide 74, however, are always removed. Synthetic yeast pre-tRNA(Phe) substrates containing the complete CCA74-76 sequence were processed with crude or partially purified chloroplast enzyme fractions. The 3'-extended substrates (tRNA-CCA-trailer) were cleaved exclusively between nucleotides 74 and 75 to give tRNA-COH, whereas a 3'-mature transcript (tRNA-CCAOH) was not cleaved at all. A 5'-, 3'-extended chloroplast tRNA-CAG-trailer was also processed entirely to tRNA-COH. Furthermore, a 5'-mature, 3'-extended yeast pre-tRNA(Phe) derivative, tRNA-ACA-trailer, in which C74 was replaced by A, was cleaved precisely after A74. In contrast, we found that a partially purified enzyme fraction (a nuclear/cytoplasmic activity) from wheat embryo cleaved the 3'-extended yeast tRNA(Phe) precursors between nucleotides 73 and 74 to give tRNA(OH). This specificity is consistent with that of all previously characterized nuclear enzyme preparations. We conclude that (i) chloroplast tRNA 3'-processing endonuclease cleaves after nucleotide 74 regardless of the nature of the surrounding sequences; (ii) this specificity differs from that of the plant nuclear/cytoplasmic processing nuclease, which cleaves after base 73; and (iii) since 3'-mature tRNA is not a substrate for either activity, these 3' nucleases must require substrates possessing a 3'-terminal extension that extends past nucleotide 76. This substrate specificity may prevent mature tRNA from counterproductive cleavage by the 3' processing system.

Kinetics of the processing of the precursor to 4·5 S RNA, a naturally occurring substrate for RNase P from Escherichia coli

A study was made of the cleavage by M1 RNA and RNase P of a non-tRNA precursor that can serve as a substrate for RNase P from Escherichia coli, namely, the precursor to 4·5 S RNA (p4·5S). The overall efficiency of cleavage of p4·5S by RNase P is similar to that of wild-type tRNA precursors. However, unlike the reaction with wild-type tRNA precursors, the reaction catalyzed by the holoenzyme with p4·5S as substrate has a much lower K m value than that catalyzed by M1 RNA with the same substrate, indicating that the protein subunit plays a crucial role in the recognition of p4·5S. A model hairpin substrate, based on the sequence of p4·5S, is cleaved with greater efficiency that the parent molecule. The 3′-terminal CCC sequence of p4·5 S may be as important for cleavage of this substrate as the 3′-terminal CCA sequence is for cleavage of tRNA precursors.

Sites of interaction of the CCA end of peptidyl-tRNA with 23S rRNA.

Oligonucleotide fragments derived from the 3' CCA terminus of acylated tRNA, such as CACCA-(AcPhe), UACCA-(AcLeu), and CAACCA-(fMet), bind specifically to ribosomes in the presence of sparsomycin and methanol [Monro, R. E., Celma, M. L. & Vazquez, D. (1969) Nature (London) 222, 356-358]. All three oligonucleotides protect a characteristic set of bases in 23S rRNA from chemical probes: G2252, G2253, A2439, A2451, U2506, and U2585. A2602 shows enhanced reactivity. These account for most of the same bases that are protected when peptidyl-tRNA analogues such as AcPhe-tRNA are bound to the ribosomal P site, and correspond precisely to those bases whose protection is abolished by removal of the 3'-CA end of tRNA. We conclude that most of the observed interactions between tRNA and 23S rRNA in the 50S ribosomal P site involve the conserved CCA terminus of tRNA. Sparsomycin may inhibit protein synthesis by stabilizing interaction between the peptidyl-CCA and the 23S P site, preventing formation of the intermediate A/P hybrid state.

The chromosomal integration site of theStreptomyces element pSAM2 overlaps a putative tRNA gene conserved among actinomycetes

The pSAM2 element of Streptomyces ambofaciens integrates site-specifically in the genome of different Streptomyces species by recombination between a 58 bp sequence common to the plasmid (attP) and the chromosome (attB). Southern hybridization analysis showed that sequences similar to the pSAM2 attB site were found in other actinomycetes (Mycobacterium, Nocardia, Micromonospora) as well as unrelated bacteria (Bacillus circulans, Escherichia coli, Clostridium botulinum, Bordetella pertussis, and Legionella pneumophila). Hybridizing fragments from B. circulans and Mycobacterium tuberculosis were cloned and sequenced. Comparison of these sequences with the sequence of the integration zone of S. ambofaciens revealed a conserved region of 76 bp which overlapped with the attB site. This conserved sequence was similar to the Salmonella typhimurium and E. coli tRNA(pro1) genes as well as a number of eucaryotic tRNA genes and had a proline-tRNA-like cloverleaf structure. Furthermore, the Streptomyces lividans attB site of the Streptomyces glaucescens element pIJ408 was also found to overlap a potential tRNA gene (tRNA(thr)). We note here that these two putative tRNA genes as well as those which overlap the attB site of the elements SLP1 of Streptomyces coelicolor and pMEA100 of Nocardia mediterranei all contain the site where integrative recombination takes place. These presumptive actinomycete tRNA genes lack the 3' terminal CCA sequence found in most procaryotic tRNA genes.

Five transfer RNA genes lacking CCA termini are clustered in the chromosome of Streptomyces rimosus

The nucleotide sequence of a 1105 bp Streptomyces rimosus DNA fragment containing five transfer RNA genes was determined. Two tRNA(Gln) (CUG) genes, differing by 1 bp in the aminoacyl stem, and three identical tRNA(Glu) (CUC) genes were identified. The five tRNA genes, arranged in the order: Gln1-Glu1-Glu2-Gln2-Glu3, were separated by short, nonhomologous intergenic regions. Surprisingly, none of these tRNA genes encoded the CCA 3' terminus of mature tRNAs. All five encoded tRNAs for the translation of GC rich codons, which are preferentially used in Streptomyces genes (CAG and GAG, respectively). We recently reported nucleotide sequences of two initiator tRNA genes from S. rimosus, which also do not encode the CCA end of mature tRNAs. It is therefore very likely that S. rimosus represents an example of those eubacteria in which the majority of tRNA genes do not encode the 3' terminal CCA end of mature tRNAs. Evolutionary implications of this finding remain to be elucidated.

Nucleotide sequences of nine tRNA genes fromMicrococcus luteus

The nucleotide sequences of nine tRNA genes located in the ACKNOWLEDGEMENT different Pstl DNA fragments from Micrococcus luteus were _, . , , , w . . , . determined (MLTR003 -MLTR011). Each tRNA gene sequence Supported by grants from the Ministry of Education, Science and is preceded by a putative promoter and followed by a probable ° a P a n terminator. The 3'-terminal CCA is coded for 4 out of 9 tRNA genes examined.

Modification of Bacillus subtilis elongation factor Tu by N-tosyl-L phenylalanyl chloromethane abolishes its ability to interact with the 3′-terminal polynucleotide structure but not with the acyl bond in aminoacyl-tRNA

Modification of B. subtilis EF-TU by N-tosyl-L-phenylalanyl chloromethane destroyed its ability to promote protein synthesis and resulted in selective dissociation of the two binding activities of the protein for aminoacyl-tRNA. The modified EF-Tu was completely ineffective in the protection of the 3′-terminal CCA structure of tRNA against pancreatic ribonuclease, while remaining almost fully active in the protection of the ester bond between the 3′-terminal adenosine and the amino acid residue in aminoacyl-tRNA.

Interaction of tRNA with 23S rRNA in the ribosomal A, P, and E sites

Three sets of conserved nucleotides in 23 rRNA are protected from chemical probes by binding of tRNA to the ribosomal A, P, and E sites, respectively. They are located almost exclusively in domain V, primarily in or adjacent to the loop identified with the peptidyl transferase function. Some of these sites are also protected by antibiotics such as chloramphenicol, which could explain how these drugs interfere with protein synthesis. Certain tRNA-dependent protections are abolished when the 3′-terminal A or CA or 2′,3′-linked acyl group is removed, providing direct evidence for the interaction of the conserved CCA terminus of tRNA with 23S rRNA. When the EF-Tu·GTP·aminoacyl-tRNA ternary complex is bound to the ribosome, no tRNA-dependent A site protections are detected in 23S rRNA until EF-Tu is released. Thus, EF-Tu prevents interaction of the 3′ terminus of the incoming aminoacyl-tRNA with the peptidyl transferase region of the ribosome during anticodon selection, thereby permitting translational proofreading.

Genes encoding 5S rRNA and tRNAs in the extremely thermophilic archaebacterium Methanothermus fervidus

Methanothermus fervidus was shown to have two 5S rRNA-encoding genes linked in rRNA operons to 16S and 23S rRNA-encoding genes. Sequencing of a cloned 5S rRNA gene confirmed that M. fervidus is a member of the Methanobacteriales, although its 5S rRNA is also similar in both primary sequence and predicted secondary structure to the 5S rRNA of the non-methanogenic, but also extremely thermophilic archaebacterium, Thermococcus celer. Two clusters of tRNA genes have also been cloned and sequenced form M. fervidus. The smaller cluster, cloned in pET5401, is composed of 5′-tRNA Thr UGU-tRNA Pro UGG-tRNA Asp GUC-tRNA Lys UUU-3′ and the larger cluster, cloned in pET5475, is composed of 5′-tRNA Asn GUU-tRNA Met CAU-tRNA Glu UUC-tRNA Leu UAG-tRNA His GUG-3′. The encoded tRNAs, with the exception of the tRNA Leu, translate abundant codons in M. fervidus. The tRNA genes do not contain introns or encode 3′-terminal CCA residues. Homologous clusters of tRNA genes have been sequenced from Methanococcus vannielii and Methanococcus voltae, so that comparisons of transcription signals, gene organizations and primary sequences can be made and features possibly related to thermostability identified. During evolution, a 5S rRNA gene appears to have been incorporated into the cluster of tRNA genes in the methanococci but not in M. fervidus.